Fluorescence spectroscopy is a highly sensitive, nearly background-free technique for chemical detection. Fluorescence detection is typically achieved by either single-line UV excitation and dispersion of the entire fluorescence emission spectrum (e.g., laser induced fluorescence (LIF)) or by tuning the excitation source over a wide wavelength range and detecting the entire spectrum of emitted light as a function of excitation wavelength with a broad band detector (e.g., excitation spectroscopy). In liquid samples excitation and emission bands are broad, making it difficult to identify an analyte. Excitation emission matrix (EEM) spectroscopy (EEMS) combines both techniques. The excitation wavelength is scanned and an EEM is produced by recording a fluorescence emission spectrum at each of the many excitation wavelengths. The EEM is used to generate a three-dimensional spectrum (resembling a “topographical map”) that allows analytes to be distinguished in a sample of a mixture by separating broad fluorescent features into key dominant spectroscopic components that may correspond to the individual fluorophores, in some cases.
Determining the concentration of specific fluorophores within a mixture can prove complicated, especially when the broad fluorescence peaks overlap. This problem can be solved using multivariate data analysis, such as principle component analysis (PCA) or parallel factor analysis (PARAFAC). Through such analyses the excitation emission matrix spectra can be decomposed into its various components and hence number of fluorophores, a feat which would be otherwise difficult for complicated mixtures.
Currently, fluorescence EEMS is typically carried out using a spectrometer that scans a pivoting grating (or other light dispersing element) so that only a small wavelength ranges passes through a slit to excite the sample. At each of the excitation wavelengths the spectrometer collects the fluorescence onto a second pivoting grating and slit to direct a selected emission wavelength to a broad band photodetector, such as an avalanche photodiode or photomultiplier. In an alternative spectrometer the second slit and photodetector are replaced with an array detector for higher data acquisition rates. Since array detectors tend to be less sensitive than broad-band photodetectors the latter is less efficient as it could be. Both designs require at least one grating that is mechanically moved. This is a time-consuming process that uses only a fraction of the light to generate the EEM spectrum (e.g., about 1/10,000 for the former and 1/100 for the latter design, depending on the required spectral resolution and spectral range) of the light to generate the EEM spectrum.
A solution to this problem is to utilise a variant of wavelength division multiplexing. If each excitation wavelength is individually modulated with a unique frequency, the entire spectrum of the light source can be used to excite the sample and all the emission collected simultaneously. The spectrum can then be obtained through demodulation of the resultant signal, for example by using a Fourier transform. However, Fourier transforms require analogue or high-bit digital modulation of light intensities and thus can be afflicted with harmonics that interfere with one another and confound the results.